Viral infection of cells triggers the secretion of interferons (IFN). This is followed by IFN mediated upregulation of a large number of IFN responsive genes. This is central to the effort to control and eliminate the infection, interferon stimulated gene 15 (ISG15) is one of the most strongly induced genes following IFN stimulation. Evidence suggests that ISG15 may be an important means by which the immune system works against viruses. ISG15 is released into the sera of human subjects injected with IFN beta, the sera of gamma HV68 virus infected mice, as well as the supernatant of cell free supernatants derived from monocytes and lymphocytes. ISG15 acts a mitogen to NK cells and modulates DC differentiation in human cell lines, iSG15 is induced by several viruses including HCMV, HSV-1, influenza B virus and gammaHV68. However, the exact role of ISG15 during viral infection remains to be characterized. This proposal aims to define the mechanisms by which ISG15 mediates the immune response during viral infection. The guiding hypothesis is that ISG15 is an IFN-induced cytokine with antiviral properties, and these properties are achieved by its effects on natural killer (NK) cells and dendritic cells (DCs). Using mouse models, this proposal aims to characterize the biochemical nature of the released forms of ISG15 by determining the size of purified ISG15 and sequencing the multiple forms of ISG15 that are released into the serum in response to viral infection and IFN stimulation. It also aims to define the effect of ISG 15 on activation, expansion and differentiation of NK cells and dendritic cells. Finally, the proposal aims to determine whether ISG15 has an antiviral effect both in vivo and in vitro during infection by gamma HV68. Antiviral effect will be determined by monitoring changes in the expression of cell surface markers as Measures of activation, as well as changes in viral titers.